Introduction to PCR
Not built yet. Lab 1 - PCR Materials Per student * 1 x plastic disposable cup * 1 x 1 mL transfer pipette * 1 x colored microtube * 200uL Instagene mix in screw top microtube Per bench * 2 x P20 or P200 micropipettors * 2 x Xcluda micropipette tips * Beaker of 0.9% Saline solution * Ice bucket * Discard Beaker * Used Tip Beaker On side or back bench * Vortexer (as many as we have) * 56 Degree water bath with microtube rack * boiling 100 Degree water bath with microtube rack * Microcentrifuge * Thermalcycler (PROGRAMMED to PV92) On Instructor's Bench (For PCR portion of the lab - keep up there so students don't confuse the supplies) * Ice bucket * PCR tubes with PCR master mix ( 1 for each table) ** Add the primers no more than an hour before the class ** Make a tube fore each table - 4? people ** Inform the instructor that if they misuse the micropipettors, there may not be enough for all students * Capless PCR tube holders Methods 0.9% Saline Solution * Prepare a 0.9% saline solution. To a 500 ml bottle of drinking water, add 4.5 grams of non-iodinated salt. Table salt is recommended. Invert the bottle until the salt goes into solution. * For each student, place 10 ml saline into a separate cup. Each student workstation should have 4 cups of saline. Aloquot InstaGene Matrix * Thoroughly mix the InstaGene matrix by gently shaking or vortexing the bottle several times to re-suspend the matrix. Be sure that the matrix is well mixed when you aliquot it. The beads settle out of solution quickly, so gently remix the bottle several times during aliquotting. * Pipet 200 µl of InstaGene matrix into each screwcap tube. Distribute one tube to each student. Each student workstation should get 4 tubes of matrix for 4 students. Master Mix with Primers Before opening any of the reagent tubes, pulse-spin the contents (~3 seconds) in a centrifuge to bring contents to the bottom of the tubes. Contents often become lodged underneath the caps during shipping.Prepare complete master mix by adding primers. For best results, the following steps should be performed within 15–30 minutes of the PCR reaction. * Pipet 1,100 µl of master mix into a labeled microcentrifuge tube. If you choose to amplify 16 student samples or less, divide the master mix into two tubes with 550 µl each. One tube will be used immediately, and the remaining master mix can be refrozen for later use. * For 32 students or 8 student workstations, label 8 microcentrifuge tubes “Master” and place the tubes on ice. * Add 22 µl of the primer mix to the 1,100 µl of master mix. (50 uL MM to 1 uL Primers) Vortex 10 seconds to mix. It is imperative that the master mix be evenly mixed after the addition of the primers. The solution should be yellow. The primers are supplied as a concentrated yellow solution in a Tris buffer. Since the primers are much more stable in a concentrated form, add the primers to the master mix just prior to beginning the laboratory exercise — not more than 30 minutes before the PCR amplification. * Aliquot 95 µl of the complete master mix into the 6 (or 7) microcentrifuge tubes labeled “Master”, supplying one tube for each student workstation (1–6). Save the remaining complete master mix for the positive control reactions. Place these tubes on ice until they will be used. Set up control PCR reactions (1 tube for every 4 groups) * Label the control PCR tubes: +/+, –/–, and +/–. * Pipet 20 µl of the +/+ template into each +/+ PCR tube. * Pipet 20 µl of the –/– template into each –/– PCR tube. * Pipet 20 µl of the +/– template into each +/– PCR tube. * Pipet 20 µl of the complete master mix into each of the control tubes. * Use a fresh tip for each tube. * Place the tubes on ice until ready to load into the thge thermalcycler. Lab 2 Gel Electrophoresis